Modified leukotoxin gene and protein

ABSTRACT

The present invention provides nucleic acid sequences encoding a modified leukotoxin protein, wherein the modification comprises the removal of nucleic acid sequences encoding amino acids within hydrophobic transmembrane domains of full length leukotoxin protein, preferably from  Mannheimia haemolytica . The modified leukotoxin proteins are useful in vaccine compositions effective against  Mannheimia haemolytica  in animals.

This application is a 371 of PCT/CA00/01498 filed Dec. 15, 2000 whichclaims benefit of 60/172,148 filed Dec. 17, 1999.

FIELD OF THE INVENTION

This invention relates to the construction and expression of a modifiedleukotoxin gene and to the use of the modified leukotoxin as a vaccine.

BACKGROUND OF THE INVENTION

Bovine pneumonic pasteurellosis, also known as shipping fever, is amajor cause of sickness and death in the feedlot cattle industry(Martin, S. W. et al. Can. J. Comp. Med. 1980, 44:1-10; Yates, W. D. G.Can. J. Comp. Med. 1982, 46:225-263). The principal microorganismassociated with this disease is Mannheimia (Pasteurella) haemolytica A1.It has been shown that M. haemolytica produces a heat labile cytotoxinwhich is specific against ruminant leukocytes (Kaehler, K. L. et al. Am.J. Vet. Res. 1980, 41:1690-1693; Shewen, P. E. et al. Infect. Immun.1982, 35:91-94). This leukotoxin has been implicated as a majorvirulence factor in the pathogenesis of M. haemolytica. Its mode ofaction has been shown to be the impairment of the primary lung defensemechanism (inactivation of alveolar macrophages) and the induction ofinflammation as a consequence of leukocyte lysis.

In the past, vaccination against M. haemolytica infection has beenattempted using both live and heat-killed bacteria of various serotypes.It has been demonstrated that vaccination with heat-killed bacteria mayactually enhance the development of pneumonia after challenge exposure(Sanford, S. E. Mod. Vet. Prac. 1984, 65:265-268). Immunization withlive M. haemolytica vaccines have generally been unsuccessful because oflow antigenicity of M. haemolytica cells and rapid inactivation by thehealthy animal (Henry, C. W. Vet. Med. 1984, 1200-1206). The cytotoxicsupernatant from M. haemolytica has also been used as a vaccine. Thispreparation contains numerous soluble antigens from the bacterium, someof which may be important in protection. Development of vaccines fromthe crude cytotoxic supernate requires the purification andcharacterization of these antigens which is difficult and costly.

Advances in molecular biology have allowed the characterization,isolation and expression of the particular genes which code for specificbacterial antigens using various recombinant DNA techniques. In fact,the gene, IktA, that codes for the full length leukotoxin (Lkt-102) ofM. haemolytica has been well characterized (Lo, R. Y. C. et al. Infect.Immun., 1987, 55:1987-1996, Lo, R. Y. C. et al. U.S. Pat. No.5,055,400). However, when the full length recombinant leukotoxin isproduced in E. coli, it is very unstable and quickly degrades. The yieldand recovery of the 102 kDa rLkt is therefore very poor, rendering thismethod of obtaining recombinant leukotoxin for use as a vaccine, costlyand inefficient.

There remains a need for a highly stable derivative of the recombinantleukotoxin that retains the antigenic and immunogenic properties of thefull length protein.

SUMMARY OF THE INVENTION

The present inventors have prepared modified leukotoxin proteins whereinthe hydrophobic transmembrane domains of the leukotoxin protein ofMannheimia haemolytica have been removed. The modified leukotoxinprotein is incapable of inserting into target cells rendering it devoidof toxic activity. Consequently, the modified protein is extremelyuseful in the preparation of vaccines. Further, the inventors have shownthat the modified leukotoxin protein is highly stable and when preparedby recombinant means is produced at several fold higher levels than fulllength leukotoxin. In addition, the modified leukotoxin protein retainsits ability to stimulate an immune response.

Accordingly, the present invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a modifiedleukotoxin protein, wherein the modification comprises the removal ofnucleic acid sequences encoding amino acids within a hydrophobictransmembrane domain of a full length leukotoxin.

In a preferred embodiment, a purified and isolated nucleic acid moleculeis provided which encodes (a) a modified leukotoxin protein as shown inFIGS. 2A and 2B or (b) a modified leukotoxin protein as shown in FIG.12.

In one embodiment, the purified and isolated nucleic acid moleculecomprises: (a) a nucleic acid sequence as shown in FIGS. 1A and 1B,wherein T can also be U; (b) nucleic acid sequences complementary to(a); (c) nucleic acid sequences which have substantial sequence homologyto (a) or (b); (d) a fragment of (a) to (c) that is at least 15 bases,preferably 20 to 30 bases, and which will hybridize to (a) to (c) understringent hybridization conditions; (e) a nucleic acid moleculediffering from any of the nucleic acids of (a) to (c) in codon sequencesdue to the degeneracy of the genetic code; or (f) a nucleic acidmolecule that is an analog of a sequence (a) to (e).

In another embodiment, the purified and isolated nucleic acid moleculecomprises: (a) a nucleic acid sequence as shown in FIG. 11, wherein Tcan also be U; (b) nucleic acid sequences complementary to (a); (c)nucleic acid sequences which have substantial sequence homology to (a)or (b); (d) a fragment of (a) to (c) that is at least 15 bases,preferably 20 to 30 bases, and which will hybridize to (a) to (c) understringent hybridization conditions; (e) a nucleic acid moleculediffering from any of the nucleic acids of (a) to (c) in codon sequencesdue to the degeneracy of the genetic code; or (f) a nucleic acidmolecule that is an analog of a sequence (a) to (e).

In another aspect, the present invention includes an expression cassettecomprising (a) a nucleic acid sequence encoding a modified leukotoxin,wherein the modification comprises the removal of nucleic acid sequencesencoding amino acids within a hydrophobic transmembrane domain of thefull length leukotoxin protein; and (b) control sequences that areoperably linked to the nucleic acid sequence whereby the nucleic acidsequence can be transcribed and translated in a host cell.

In a further aspect, the present invention provides a plasmid comprisinga nucleic acid sequence encoding a modified leukotoxin protein, whereinthe modification comprises the removal of nucleic acid sequencesencoding amino acids within a hydrophobic transmembrane domain of thefull length leukotoxin protein.

The present invention further involves host cells and microorganismstransformed with a construct or plasmid of the invention.

There is also provided a method for producing a recombinant modifiedleukotoxin protein, wherein the modification comprises the removal ofamino acids within a hydrophobic transmembrane domain of the full lengthleukotoxin protein, comprising the steps of:

(a) transforming a host cell with a nucleotide sequence of theinvention;

(b) culturing the transformed host cell under suitable conditions toproduce the modified leukotoxin; and

(c) isolating the modified leukotoxin protein.

The invention also includes a method for the production of a modifiedleukotoxin in a host cell comprising:

a) introducing into the host cell a chimeric nucleic acid sequencemolecule comprising in the 5′ to 3′ direction of transcription:

-   -   1) a first nucleic acid sequence capable of regulating        transcription in said host cell operatively linked to;    -   2) a second nucleic acid sequence encoding a modified leukotoxin        protein operatively linked to;    -   3) a third nucleic acid sequence capable of terminating        transcription in said host cell; and    -   b) culturing said host cell under suitable conditions to allow        said cell to express the modified leukotoxin protein.

In one embodiment, the host cell is a bacteria. In another embodiment,the host cell is a plant.

Further, the present invention provides a method for producing arecombinant modified leukotoxin protein as described above, wherein themodified leukotoxin protein has the amino acid sequence shown in FIGS.2A and 2B or as shown in FIG. 12, or a homolog, analog, derivative orfragment thereof.

The present invention also provides a purified and isolated polypeptidehaving an amino acid sequence of a modified leukotoxin protein,preferably having the amino acid sequence shown in FIGS. 2A and 2B or asshown in FIG. 12, or a homolog, analog, derivative or fragment thereof.

The present invention extends to cover polyclonal and monoclonalantibodies raised to a modified leukotoxin, wherein the modificationcomprises the removal of amino acids within a hydrophobic transmembranedomain of a full length leukotoxin protein, or a modified leukotoxinthat is a homolog, analog, derivative or fragment thereof.

The present invention is also directed to a vaccine compositioncomprising a pharmaceutically acceptable carrier and a modifiedleukotoxin, wherein the modification comprises the removal of aminoacids within a hydrophobic transmembrane domain of the full lengthleukotoxin protein, or a homolog, analog, derivative or fragmentthereof.

The present invention also involves a vaccine composition comprising apharmaceutically acceptable carrier and a nucleic acid sequence encodinga modified leukotoxin protein, wherein the modification comprises theremoval of sequences encoding amino acids within a hydrophobictransmembrane domain of the full length leukotoxin protein, or a nucleicacid sequence encoding a homolog, analog, derivative or fragment of themodified leukotoxin protein.

In still another aspect of the present invention, there are providedmethods for preventing or treating an infection associated with aleukotoxin such as respiratory disease in an animal comprisingadministering an effective amount of a modified leukotoxin gene orprotein of the invention to an animal in need thereof.

In still another aspect of the present invention, there are providedmethods for preventing or treating a Mannheimia infection, preferably aMannheimia haemolytica infection in an animal comprising administeringan effective amount of a modified leukotoxin gene or protein of theinvention to an animal in need thereof.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present invention will become more apparent from thefollowing description in which reference is made to the appendeddrawings in which:

FIGS. 1A and 1B (SEQ.ID.NO.:1) shows the nucleic acid sequence codingfor a modified leukotoxin (Ikt66) of the invention.

FIGS. 2A and 2B (SEQ.ID.NO.:2) shows the shows the amino acid sequencefor a modified leukotoxin (Ikt66) of the invention.

FIG. 3 is a Kyte-Doolittle hydropathy plot of the full length leukotoxinprotein showing the hydrophobic (positive) and hydrophilic (negative)regions.

FIG. 4 is a Coomassie stained SDS-PAGE where lane #3 is from theexpression of the full length Lkt-102 in E. coli, lane #4 is from theexpression of the modified Lkt-66 in E. coli and lane #5 is a negativesample where no Lkt protein was present.

FIG. 5 is a Western immunoblot of a duplicate set of proteinpreparations immunostained with rabbit anti-Lkt102 (#3) and anti-Lkt66(#4).

FIG. 6 shows the transient expression of chimeric genes in tobacco. (A)Protein extracted from tobacco leaves infiltrated with Agrobacterium,transformed with constructs containing promoterless modified greenfluorescent protein mGFP5 (lane 1), 35S-mGFP5 (lane 2) or35S-Lkt50-mGFP5 (lane 3), were blotted and probed with rabbit anti-Lkt66antiserum. The resulting Western immunoblot is shown. A cross-reactingband was observed only in lane 3 where the presence of theLkt50-containing fusion protein was expected. (B) The expression ofLkt50-mGFP5 (lane 1) and mGFP5-Lkt50(lane 2) was analyzed by Westernimmunoblot with the rabbit anti-Lkt66 antiserum as above. Agrobacteriumtransformed with vectors containing either 35S-Lkt50-mGFP5 (lane 1) or35S-mGFP5-Lkt50(lane 2) was used for infiltration. Only in the casewhere mGFP5 was fused to the C-terminus of the M. haemolytica A1Lkt50(lane 1) was fusion protein expression detected. Migration of themolecular weight markers are indicated on the right.

FIG. 7 shows the laser confocal microscopy of transgenic white cloverexpressing Lkt50-mGFP5. A section of clover leaf was mounted in waterand observed by confocal microscopy. Images from two channels (red forchlorophyll fluorescence and green for mGFP5 fluorescence) were mergedto produce the micrographs shown. Leaves from untransformed clover (A)do not exhibit the green fluorescence which is present in transgenicclover expressing Lkt50-mGFP5 (B). The pattern of green fluorescence isconsistent with an ER localization. The bar indicates 100 μm. Vacuoles(V), nuclei (N), and chloroplasts (Ch) are indicated in panel 8.

FIG. 8 shows the expression of Lkt50-mGFP5 in transgenic white clover.Expression of Lkt50-mGFP5 in transgenic white clover was analyzed byWestern immunoblot. Duplicate blots of proteins extracted from onetransgenic line were immunostained with either rabbit anti-Lkt66antiserum (A, lane 1) or rabbit anti-GFP monoclonal (Clonetech) (B).Molecular sizes of the pre-stained SDS-PAGE standards (Bio-Rad) (A andB, lanes M) are indicated at the left. Both antibodies detected aprotein of similar size, providing evidence that a Lkt50-mGFP5 fusionprotein was indeed produced by the transgenic clover. In addition, thesize of the fusion protein observed was close to the predicted size of79 kDa as predicted from the nucleotide sequence. In panel C, thestability of Lkt50-mGFP5 recovered from clover was examined. Proteinextracts were prepared from fresh transgenic clover (lane 2) or fromclover dried for 1 day, 2 days, 3 days, or 4 days (lanes 3-6,respectively) were analyzed by Western immunoblot. The blot was probedwith the rabbit anti-Lkt66 antiserum. A sample of M. haemolytica A1supernatant containing full-length authentic Lkt was loaded in lane 1.Migration of molecular size markers (lane M) are shown on the left.After 4 days of drying at ambient temperatures, there does not appear tobe significant degradation of the Lkt50-mGFP5 fusion protein.

FIG. 9 shows the partial purification of Lkt50-mGFP5 for immunization. Asupernatant was prepared from transgenic white clover and fractionatedby chromatofocusing (Pharmacia). Column fractions were analyzed bySDS-PAGE (A) and Western immunoblot (B). The fraction numbers areindicated on the top and size markers (lanes M) are indicated at theleft. These results show that Lkt50-mGFP5 (fractions 6, 7 and 8) can beseparated from Rubisco (strongly staining band migrating at around 56kDa) and other high molecular weight material (fractions 5 and 6).

FIG. 10 shows the immunogenicity of Lkt50-mGF5 produced by white clover.(A) Rabbits (duplicate rabbits used for each treatment) weremock-immunized with saline and adjuvant (lanes 1 and 2) or immunizedwith chromatographic fractions enriched in Lkt50-mGFP5 (lanes 3 and 4)or a saline extract from transgenic clover (lanes 5 and 6). Immune serawere used to probe a total M. haemolytica A1 protein preparation blottedonto nitrocellulose membrane. The rabbit anti-Lkt-66 antiserum (lane 7)was used as positive control. Immune serum from all four rabbitsimmunized with Lkt50-mGFP5-containing fractions recognized a bandmigrating identically with that immunostained with anti-Lkt66 (lanes3-7). Immune serum used in panel A lane 6 (rabbit 41) was analyzed tosee if it cross reacts with wild type GFP (B and C). Triplicate blots(B,C and D) were prepared containing M. haemolytica A1 total proteinpreparation (lanes 1) and purified GFP (Clontech, lanes 2). Anti-GFPantibodies (B), anti-Lkt66 (C) and rabbit 41 immune serum (used in panelA, lane 6) (D) were used to probe the membranes. Rabbit 41 serum wasable to detect mGFP5 (D, lane 2). These results suggest that the immuneserum contain antibodies directed to both Lkt50(A, lane 6 and D, lane 1)and GFP (D lane 2). Molecular size markers (B, C and D, lanes M) are asindicated on the left of panel B.

FIG. 11 (SEQ.ID.NO.:3) shows the nucleic acid sequence coding for amodified leukotoxin (Ikt50) of the invention.

FIG. 12 (SEQ.ID.NO.:4) shows the shows the amino acid sequence for amodified leukotoxin (Ikt50) of the invention.

FIG. 13 (SEQ.ID.NO.:5) shows the amino acid sequence of the full lengthleukotoxin protein from M. haemolytica.

DETAILED DESCRIPTION OF THE INVENTION

The following standard abbreviations for the amino acid residues areused throughout the specification: A, Ala—alanine; C, Cys—cysteine; D,Asp—aspartic acid; E, Glu—glutamic acid; F, Phe—phenylalanine; G,Gly—glycine; H, His—histidine; I, Ile—isoleucine; K, Lys—lysine; L,Leu—leucine; M, Met—methionine; N, Asn—asparagine; P, Pro—proline; Q,Gln—glutamine; R, Arg—arginine; S, Ser—serine; T, Thr—threonine; V,Val—valine; W, Trp—tryptophan; Y, Tyr—tyrosine; and p.Y.,P.Tyr—phosphotyrosine.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology and immunology which are known to thoseskilled in the art. Such techniques are explained fully in theliterature. See for example the following references:

1. Maniatis, Fritsch and Sambrook, Molecular Cloning: A LaboratoryManual, 1982;

2. DNA Cloning, Vol. I and II, Glover, D. N., ed. 1985;

3. Oligonucleotide Synthesis, Gait, M. J., ed. 1984;

4. Nucleic Acid Hybridization, Hames, B. D. and Higgins, S. J. eds.1986;

5. Animal Cell Culture, Freshney, R. K. ed. 1986;

6. Immobilized Cells and Enzymes, IRL Press, 1986;

7. Perbal, B. A Practical Guide to Molecular Cloning, 1984;

8. The Series, Methods In Enzymology, Colowick, S. and Naplan, N. eds.Academic Press Inc.;

9. Handbook of Experimental Immunology, Vol. I-IV, Weir, D. M andBlackwell, C. C. eds. 1986, Blackwell Scientific Publications.

I. Nucleic Acid Molecules of the Invention

The present invention includes a modified leukotoxin gene for thepreparation of a modified leukotoxin protein.

The term “modified leukotoxin gene” as used herein means a nucleic acidsequence encoding a leukotoxin protein that has been modified to removenucleic acid sequences that encode amino acids within a hydrophobictransmembrane domain of a full length leukotoxin protein.

The term “modified leukotoxin protein” means a leukotoxin protein thathas been modified to remove amino acid sequences within a hydrophobictransmembrane domain of the full length leukotoxin. A modifiedleukotoxin can have amino acids deleted in one or more of thehydrophobic domains of the full length protein.

In one embodiment, the modified leukotoxin is derived from the fulllength or naturally occurring leukotoxin from M. haemolytica. Thesequence of the full length leukotoxin protein of M. haemolytica isshown in FIG. 13 (SEQ.ID.NO.:5). Three hydrophobic domain regions arepresent in the sequence and are found at the following positions: domain1—amino acids 230-250; domain 2—amino acids 280-320; and domain 3—aminoacids 360-400. In one embodiment, the modified leukotoxin protein has adeletion of at least one of the hydrophobic domains, more preferably atleast two of the hydrophobic domains and most preferably all three ofthe hydrophobic domains.

The invention also includes modified leukotoxin proteins based onleukotoxins from other species including Actinobacillusactinomycetemcomitans (GenBank Accession nos. A37205, AAA21922, CM34731,CM34730, P16462); Pasteurella suis (U.S. Pat. No. 5,559,008);Synecnocystis sp. (BAA18765); and other P. haemolytica serotypes (T10:A35254, P55117; A11: P55118; T3: P55116; and 5943B: P55123). Thehydrophobic domains of a leukotoxin can be determined by one of skill inthe art for example by comparing the sequence to the leukotoxin of M.haemolytica shown in FIG. 13 and matching the hydrophobic regions or bypreparing a Kyte-Doolittle hydropathy plot of the leukotoxin protein. Insome instances, the hydrophobic domains are identified in the publishedsequences. For example, the hydrophobic regions of A.actinomycetemcomitans may be found in the above referenced GenBankAccession nos. or in J. Biol. Chem. 264(26), 15451-15456, 1989.

The modification or deletion in the modified leukotoxin protein shouldbe sufficient to render the modified protein incapable of inserting intothe membrane of target cells rendering it devoid of toxic activity.

Preferably, the modified leukotoxin has at least 20 amino acids, morepreferably from 50 to 500 amino acids deleted from one or morehydrophobic domains. The modified leukotoxin protein may additionallyhave deletions in other portions of the protein such as in the Nterminus or C terminus outside of the hydrophobic domains.

Advantageously, the modified leukotoxin protein when prepared byrecombinant means is produced at levels that are higher than when thefull length leukotoxin protein is prepared under the same conditions.The modified leukotoxin protein retains its ability to generate animmune response. In particular, the modified leukotoxin protein caninduce an antibody response when used to immunize an animal.

Generally, a modified leukotoxin gene can be produced by an in-framedeletion of amino acids within the hydrophobic transmembrane domains ofthe full length leukotoxin gene, IktA. Accordingly, the presentinvention provides a purified and isolated nucleic acid moleculecomprising a sequence encoding a modified leukotoxin wherein themodification comprises the removal of nucleic acid sequences encodingamino acids within at least one transmembrane domain of the full lengthleukotoxin gene.

The term “isolated” refers to a nucleic acid substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors, or other chemicals when chemicallysynthesized

The term “nucleic acid sequence” refers to a sequence of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars andintersugar (backbone) linkages. The term also includes modified orsubstituted sequences comprising non-naturally occurring monomers orportions thereof, which function similarly. The nucleic acid sequencesof the present invention may be ribonucleic (RNA) or deoxyribonucleicacids (DNA) and may contain naturally occurring bases including adenine,guanine, cytosine, thymidine and uracil. The sequences may also containmodified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl,2-propyl, and other alkyl adenines, 5-halo uracil, 5-halo cytosine,6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil,4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol adenine,8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkylguanines, 8-hydroxyl guanine and other 8-substituted guanines, other azaand deaza uracils, thymidines, cytosines, adenines, or guanines,5-trifluoromethyl uracil and 5-trifluoro cytosine.

In one embodiment, the inventors have prepared a modified leukotoxinprotein by deleting the nucleic acid sequences that encode the entireregion containing the three hydrophobic domains in the leukotoxin fromM. haemolytica. The hydrophobic transmembrane domains, spanning fromabout amino acid number 50 to about amino acid number 400, of the fulllength leukotoxin from M. haemolytica are clearly visible in theKyte-Doolittle hydropathy plot shown in FIG. 3. The in-frame deletionmay be performed by the digestion of IktA using the appropriaterestriction enzyme or enzymes and re-ligation of the two external piecesusing standard techniques. The nucleic acids coding for the hydrophobicdomains of leukotoxin are located approximately between about position618 and about position 1653 in the IktA sequence (see Lo, R. Y. C. etal. Infect. and Immun. 1987, 55:1987-1996). The use of any restrictionenzyme or enzymes that result in the removal of this region in lkta iswithin the scope of the present invention. In a preferred embodiment ofthe invention, the restriction enzyme is Nael. After deletion of thein-frame fragment, the remaining IktA gene (named IktAΔN) codes for amodified protein of approximately 66 kDa, hence the name Lkt-66. Thenucleic acid sequence of a modified leukotoxin gene obtained using Naeland the corresponding amino acid sequence of the modified leukotoxin areshown in FIGS. 1A and 1B (or SEQ.ID.NO.:1) and FIGS. 2A and 2B (orSEQ.ID.NO.:2), respectively. The invention extends to cover nucleic acidand amino acid sequences substantially homologous and functionallyequivalent to those shown in FIGS. 1A and 1B and FIGS. 2A and 2B,respectively as well as analogs, derivatives and fragments thereof.

Accordingly, the present invention provides an isolated nucleic acidmolecule having a sequence which encodes a modified leukotoxin having anamino acid sequence as shown in FIGS. 2A and 2B (or SEQ.ID.NO.:2).

Preferably, the purified and isolated nucleic acid molecule comprises:

(a) a nucleic acid sequence as shown in FIGS. 1A and 1B (orSEQ.ID.NO.:1), wherein T can also be U;

(b) a nucleic acid sequences complementary to (a);

(c) a nucleic acid sequence which has substantial sequence homology to(a) or (b);

(d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to30 bases, and which will hybridize to (a) to (c) under stringenthybridization conditions;

(e) a nucleic acid molecule differing from any of the nucleic acids of(a) to (c) in codon sequences due to the degeneracy of the genetic code;or

(f) a nucleic acid sequence that is an analog of a sequence (a) to (e).

In another embodiment, the inventors have prepared a modified leukotoxinprotein wherein the N-terminal end (up until about amino acid number450) and 52 amino acids from the C-terminal end of the full lengthleukotoxin protein from M. haemolytica, have been deleted. The deletionin the N-terminal end includes the 3 hydrophobic domains of theleukotoxin as illustrated in FIG. 13 (SEQ.ID.NO.:5). This modifiedleukotoxin protein is termed Lkt 50 and its preparation is more fullydescribed in Example 2. The nucleic acid sequence of the gene encodingLkt 50 is shown in FIG. 11. The amino acid sequence of Lkt 50 is shownin FIG. 12 (SEQ.ID.NO.:4).

Accordingly, the present invention provides an isolated nucleic acidmolecule having a sequence which encodes a modified leukotoxin having anamino acid sequence as shown in FIG. 12 (SEQ.ID.NO.:4).

Preferably, the purified and isolated nucleic acid molecule comprises:

(a) a nucleic acid sequence as shown in FIG. 11 (SEQ.ID.NO.:3), whereinT can also be U;

(b) a nucleic acid sequences complementary to (a);

(c) a nucleic acid sequence which has substantial sequence homology to(a) or (b);

(d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to30 bases, and which will hybridize to (a) to (c) under stringenthybridization conditions;

(e) a nucleic acid molecule differing from any of the nucleic acids of(a) to (c) in codon sequences due to the degeneracy of the genetic code;or

(f) a nucleic acid sequence that is an analog of a sequence (a) to (e).

It will be appreciated that the invention also includes nucleic acidmolecules encoding homologs, analogs, derivatives and fragments ofmodified leukotoxin proteins of the invention wherein such homologs,analogs, derivatives and fragments have the same utility as the modifiedleukotoxin proteins.

In particular, the invention includes nucleic acid molecules comprisingnucleic acid sequences having substantial sequence homology with thenucleic acid sequences as shown in FIGS. 1A and 1B or 11 and fragmentsthereof. The term “sequences having substantial sequence homology” meansthose nucleic acid sequences which have slight or inconsequentialsequence variations from these sequences, i.e., the sequences functionin substantially the same manner to produce functionally equivalentproteins. The variations may be attributable to local mutations orstructural modifications.

Two nucleotide sequences are “substantially homologous” when at leastabout 80% preferably at least about 90% and most preferably at leastabout 95% of the nucleotides or amino acids match over a defined lengthof the molecule. Nucleotide sequences that are substantially homologouscan be identified in a Southern hybridization experiment under, forexample, stringent hybridization conditions, as defined below.

Another aspect of the invention provides a nucleic acid molecule, andfragments thereof having at least 15 bases, which hybridize to nucleicacid molecules of the invention under hybridization conditions,preferably stringent hybridization conditions. Appropriate stringencyconditions which promote DNA hybridization are known to those skilled inthe art, or may be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following maybe employed: 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C. The stringency may be selectedbased on the conditions used in the wash step. For example, the saltconcentration in the wash step can be selected from a high stringency ofabout 0.2×SSC at 50° C. In addition, the temperature in the wash stepcan be at high stringency conditions, at about 65° C.

Isolated and purified nucleic acid molecules having sequences whichdiffer from the nucleic acid sequence shown in FIGS. 1A and 1B or 11 dueto degeneracy in the genetic code are also within the scope of theinvention. Such nucleic acids encode functionally equivalent proteinsbut differ in sequence from the above mentioned sequences due todegeneracy in the genetic code.

The term “a nucleic acid sequence which is an analog” means a nucleicacid sequence which has been modified as compared to the sequence ofFIGS. 1A and 1B or 11 wherein the modification does not alter theutility of the sequence (i.e. does not insert into target cells and isuseful in vaccine formulations) as described herein. The modifiedsequence or analog may have improved properties over the sequence shownin FIGS. 1A and 1B or 11. One example of a modification to prepare ananalog is to replace one of the naturally occurring bases (i.e. adenine,guanine, cytosine or thymidine) of the sequence shown in FIGS. 1A and 1Bor FIG. 11 with a modified base such as such as xanthine, hypoxanthine,2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halouracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine,pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thioladenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiolguanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Another example of a modification is to include modified phosphorous oroxygen heteroatoms in the phosphate backbone, short chain alkyl orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages in the nucleic acid molecule shown inFIGS. 1A and 1B or FIG. 11. For example, the nucleic acid sequences maycontain phosphorothioates, phosphotriesters, methyl phosphonates, andphosphorodithioates.

A further example of an analog of a nucleic acid molecule of theinvention is a peptide nucleic acid (PNA) wherein the deoxyribose (orribose) phosphate backbone in the DNA (or RNA), is replaced with apolyamide backbone which is similar to that found in peptides (P. E.Nielsen, et al Science 1991, 254, 1497). PNA analogs have been shown tobe resistant to degradation by enzymes and to have extended lives invivo and in vitro. PNAs also bind stronger to a complimentary DNAsequence due to the lack of charge repulsion between the PNA strand andthe DNA strand. Other nucleic acid analogs may contain nucleotidescontaining polymer backbones, cyclic backbones, or acyclic backbones.For example, the nucleotides may have morpholino backbone structures(U.S. Pat. No. 5,034,506). The analogs may also contain groups such asreporter groups, a group for improving the pharmacokinetic orpharmacodynamic properties of nucleic acid sequence.

A nucleic acid molecule of the invention may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

Determination of whether a particular nucleic acid molecule encodes anovel protein of the invention may be accomplished by expressing thecDNA in an appropriate host cell by standard techniques, and testing theactivity of the protein using the methods as described herein. A cDNAhaving the activity of a novel protein of the invention so isolated canbe sequenced by standard techniques, such as dideoxynucleotide chaintermination or Maxam-Gilbert chemical sequencing, to determine thenucleic acid sequence and the predicted amino acid sequence of theencoded protein.

II. Novel Proteins of the Invention

The invention further broadly contemplates an isolated modifiedleukotoxin protein wherein the modification comprises the removal ofamino acids within a hydrophobic transmembrane domain of a full lengthleukotoxin protein. The modified leukotoxin is incapable of insertinginto target membranes making it very useful in the preparation of avaccine. The inventors have surprisingly shown that the modifiedleukotoxin protein of the invention is much more stable than the fulllength leukotoxin protein. When prepared using recombinant DNAtechniques, the yield of the modified leukotoxin protein is at least 50times higher than that of the full length leukotoxin protein.

In one embodiment of the invention, an isolated protein is providedwhich has the amino acid sequence as shown in FIGS. 2A and 2B.

In another embodiment of the invention, an isolated protein is providedwhich has the amino acid sequence as shown in FIG. 12.

Within the context of the present invention, a protein of the inventionmay include various structural forms of the primary protein which remainimmunogenic. For example, a protein of the invention may be in the formof acidic or basic salts or in neutral form. In addition, individualamino acid residues may be modified by oxidation or reduction.

In addition to the amino acid sequence (FIGS. 2A and 2B or FIG. 12), theprotein of the present invention may also include analogs, homologs,derivative and fragments of the modified leukotoxin proteins asdescribed herein.

Analogs of the protein having the amino acid sequence shown in FIGS. 2Aand 2B or FIG. 12, may include, but are not limited to an amino acidsequence containing one or more amino acid substitutions, insertions,and/or deletions. Amino acid substitutions may be of a conserved ornon-conserved nature. Conserved amino acid substitutions involvereplacing one or more amino acids of the proteins of the invention withamino acids of similar charge, size, and/or hydrophobicitycharacteristics. When only conserved substitutions are made theresulting analog should be functionally equivalent. Non-conservedsubstitutions involve replacing one or more amino acids of the aminoacid sequence with one or more amino acids which possess dissimilarcharge, size, and/or hydrophobicity characteristics.

One or more amino acid insertions may be introduced into the amino acidsequences shown in FIGS. 2A and 2B or FIG. 12. Amino acid insertions mayconsist of single amino acid residues or sequential amino acids rangingfrom 2 to 15 amino acids in length. For example, amino acid insertionsmay be used to enhance the immunogenicity of the protein.

Deletions may consist of the removal of one or more amino acids, ordiscrete portions from the amino acid sequence shown in FIGS. 2A and 2Bor FIG. 12. The deleted amino acids may or may not be contiguous.

The lower limit length of the resulting analog with a deletion mutationis about 10 amino acids, preferably 100 amino acids.

Analogs of a protein of the invention may be prepared by introducingmutations in the nucleotide sequence encoding the protein. Mutations innucleotide sequences constructed for expression of analogs of a proteinof the invention must preserve the reading frame of the codingsequences.

Furthermore, the mutations will preferably not create complementaryregions that could hybridize to produce secondary mRNA structures, suchas loops or hairpins, which could adversely affect translation of thereceptor mRNA.

Mutations may be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site specific mutagenesisprocedures may be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired.

Deletion of a protein of the invention may also be constructed byutilizing convenient restriction endonuclease sites adjacent to thedesired deletion. Subsequent to restriction, overhangs may be filled in,and the DNA religated. Exemplary methods of making the alterations setforth above are disclosed by Sambrook et al (Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989).

The proteins of the invention also include homologs of the amino acidsequence shown in FIGS. 2A and 2B or FIG. 12 as described herein. Suchhomologs are proteins whose amino acid sequences are comprised of aminoacid sequences that hybridize under stringent hybridization conditions(see discussion of stringent hybridization conditions herein) with aprobe used to obtain a protein of the invention. Homologs of a proteinof the invention will have the same regions which are characteristic ofthe protein. A homologous protein includes a protein with an amino acidsequence having at least 80%, preferably 90%, most preferably 95%identity with the amino acid sequence as shown in FIGS. 2A and 2B orFIG. 12.

The invention also contemplates isoforms of the proteins of theinvention. An isoform contains the same number and kinds of amino acidsas a protein of the invention, but the isoform has a different molecularstructure. The isoforms contemplated by the present invention are thosehaving the same properties as a protein of the invention as describedherein.

The present invention also includes derivatives of the proteins of theinvention. “Derivative” refers to a peptide having one or more residueschemically derivatized by reaction of a functional side group. Suchderivatized molecules include for example, those molecules in which freeamino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included asderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite activity ismaintained.

The present invention also includes a protein of the inventionconjugated with a selected protein, or a selectable marker protein toproduce fusion proteins.

The proteins of the invention (including homologs, analogs, etc.) may beprepared using recombinant DNA methods. In particular, nucleic acidmolecules of the present invention having a sequence which encodes amodified leukotoxin protein of the invention may be incorporatedaccording to procedures known in the art into an appropriate expressionvector or replicon which ensures good expression of the protein.Accordingly, the invention provides a method for the production of amodified leukotoxin in a host cell comprising:

a) introducing into the host cell a chimeric nucleic acid sequencemolecule comprising in the 5′ to 3′ direction of transcription:

-   -   1) a first nucleic acid sequence capable of regulating        transcription in said host cell operatively linked to;    -   2) a second nucleic acid sequence encoding a modified leukotoxin        protein operatively linked to;    -   3) a third nucleic acid sequence capable of terminating        transcription in said host cell; and

b) culturing said host cell under suitable conditions to allow said cellto express the modified leukotoxin protein.

The chimeric nucleic acid is prepared in a vector. Numerous cloningvectors are known to those skilled in the art and the selection of anappropriate cloning vector is a matter of choice. The term vector ismeant to mean a replicon, such as a plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. Examples of recombinant DNA vectors forcloning, and host cells which they can transform, include thebacteriophage lambda (E. coli), pTTQ18 (E. coli), pBR322 (E. coli),pACYC177 (E. coli), pKT230 (gram negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), Ycp19 (Saccharomyces), bovine papilloma virus(mammalian cells) and pBI121 (plant).

The coding sequence for the modified leukotoxin can be placed under thecontrol of a promoter, ribosome binding site (for bacterial expression)and, optionally, an operator (collectively referred to herein as“control elements”), so that the DNA sequence encoding the protein istranscribed into mRNA in the host cell transformed by a vectorcontaining this expression construction. The coding sequence may or maynot contain a signal peptide or a leader sequence. In one embodiment,the expression of modified gene is regulated by the inducible tacpromoter.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the bacterialantigen sequences relative to the growth of the host cell. Regulatorysequences are known to those skilled in the art, and examples includethose which cause the expression of a gene to be turned on or off inresponse to a chemical or physical stimulus, including the presence of aregulatory compound. Other types of regulatory elements may be presentin the vector, for example, enhancer sequences. The subject proteins mayalso be expressed in the form of a fusion protein, wherein aheterologous amino acid sequence is expressed at the N-terminal orC-terminal.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the control of the control sequences (i.e. RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the sequences encodingthe particular protein of interest may be desirable to achieve this end.For example, in some cases it may be necessary to modify the sequence sothat it may be attached to the control sequences with the appropriateorientation, i.e., to maintain the reading frame. The control sequencesand other regulatory sequences may be ligated to the coding sequenceprior to insertion into a vector, such as the cloning vectors describedabove. Alternatively, the coding sequence can by cloned directly into anexpression vector which already contains the control sequences and anappropriate restriction site.

In some cases, it may be desirable to add sequences which cause thesecretion of the protein from the host organism. Such secretionsequences may be located in the same vector or in a separate vector.When E. coli is the host, the use of a separate vector, for example, theplasmid pWAM716, which codes for the hIyB/D secretion functions, ispreferred.

Depending on the expression system and the host selected, the protein ofthe present invention may be produced by growing host cells, transformedby an expression vector described above, under conditions whereby theprotein of interest is expressed. The protein may be then isolated fromthe host cells and purified. If the expression system secretes theprotein into the growth media, the protein is purified directly from themedia. If the protein is not secreted, it is isolated from cell lysates.The selection of the appropriate growth conditions and recovery methodsare within the skill of the art.

The host cell may be selected from a wide range of host cells includingplants, bacteria, yeasts, insects and mammals. In one embodiment thehost cell is a plant cell. The plant may be selected from various plantfamilies including Brassicaceae, Compositae, Euphorbiaceae, Leguminosae,Linaceae, Malvaceae, Umbilliferae, Graminae, Nicotiana and Trifoliumspp. Particular types of plants that may be used to prepare the modifiedleukotoxin protein include tobacco (Nicotiana tobacum), white clover(Trifolium repens), soybean (Glycine max), rapeseed (Brassica napus,Brassica campestris), sunflower (Helianthus annuus), cotton (Gossypiumhirsutum), corn (Zea mays), alfalafa (Medicago sativa), wheat (Triticumsp.), barley (Hordeum vulgare), oats (Avena sativa L.), sorghum (Sorghumbicolor), Arabidopsis thaliana, potato (Solanum sp.), flax/linseed(Linum usitatissimum), safflower (Carthamus tinctorius), oil palm(Eleais guineeis), groundnut (Arachis hypogaea), Brazil nut(Bertholletia excelsa) coconut (Cocus nucifera), castor (Ricinuscommunis), coriander (Coriandrum sativum), squash (Cucurbita maxima),jojoba (Simmondsia chinensis) and rice (Oryza sativa).

Accordingly, the invention provides a method for the production of amodified leukotoxin in a plant comprising:

a) introducing into a plant cell a chimeric nucleic acid sequencemolecule comprising in the 5′ to 3′ direction of transcription:

-   -   1) a first nucleic acid sequence capable of regulating        transcription in said plant cell operatively linked to;    -   2) a second nucleic acid sequence encoding a modified leukotoxin        protein operatively linked to;    -   3) a third nucleic acid sequence capable of terminating        transcription in said plant cell; and

b) growing said plant cell into a mature plant wherein said plantexpresses the modified leukotoxin protein.

The preparation of the modified leukotoxin protein in plants offers asignificant advantage as the plant can be consumed directly by theanimal as a vaccine. The modified leukotoxin does not necessarily haveto be isolated from the plant.

The proteins of the present invention may also be produced by chemicalsynthesis such as solid phase peptide synthesis. Such methods are knownto those of skill in the art as discussed above for the synthesis ofnucleic acids.

The proteins of the present invention (or homologs, analogs, derivativesand fragments thereof may be used to produce both polyclonal ormonoclonal antibodies. Antibodies that bind a protein of the inventionand its homologs can be prepared using techniques known in the art suchas those described by Kohler and Milstein, Nature, 1975, 256:495 and inU.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439 and 4,411,993. (See alsoMonoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennett, McKearn and Bechtol, eds. Plenum Press, 1980 andAntibodiesL A Laboratory Manual, Harlow and Lane, eds. Cold SpringHarbor Laboratory Press, 1988).

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies, polyclonal antibodies, antibodyfragments (e.g. Fab and F(ab( )₂) and recombinantly produced bindingpartners. Antibodies are understood to be reactive against the proteinencoded by the modified leukotoxin gene of M. haemolytica and itshomologs if they bind to the receptor with an affinity of greater thanor equal to 10⁻⁶ M.

Polyclonal antibodies may be readily generated by one of ordinary skillin the art from a variety of warm-blooded animals such as horses, cows,various fowl, rabbit, mice or rats. Briefly, a modified leukotoxinprotein or a homolog, analog, derivative or fragment thereof, may beused through intraperitoneal, intramuscular, intraocular, orsubcutaneous injections, in conjunction with an adjuvant such asaluminum hydroxide or saponin. Following several booster immunizations,samples of serum are collected and tested for reactivity to the protein.Once the titer of the animal has reached a plateau in terms ofreactivity to the receptor protein, larger quantities of antisera may beobtained by weekly bleedings, or by exsanguinating the animal.

Monoclonal antibodies may also be generated using conventionaltechniques. Generally, hybridoma cell lines are prepared by a processinvolving the fusion under appropriate conditions of an immortalizingcell line and spleen cells from an animal appropriately immunized toproduce the desired antibody. Immortalizing cell lines may be murine inorigin however, cell lines of other mammalian species may be employedincluding those of rat, bovine, canine, human origin and the like. Theimmortalizing cell lines are most often of tumor origin, particularlymyeloma cells, but may also include normal cells transformed with, forexample, Epstein Barr Virus. Any immortalizing cell may be used toprepare the hybridomas of the present invention.

Antibody producing cells may be employed as fusion partners such asspleen cells or peripheral blood lymphocytes. The animal from which thecells are to be derived may be immunized at intervals with a protein ofthe invention or its homolog.

The immortalizing cells and lymphoid cells may be fused to formhybridomas according to standard and well-known techniques employingpolyethylene glycol as a fusing agent. Alternatively, fusion may beaccomplished by electrofusion.

Hybridomas are screened for appropriate monoclonal antibody secretion byassaying the supernatant or protein purified from the ascites forreactivity with a protein of the invention or its homolog.

The monoclonal antibodies produced by the hybridoma cell lines of theinvention are also part of the present invention. It is understood thatimmunoglobulins may exist in acidic, basic or neutral form depending ontheir amino acid composition and environment, and they be found inassociation with other molecules such as saccharides or lipids. Themonoclonal antibodies produced by hybridoma cell lines of the inventionmay be directed to one or more epitope of a modified leukotoxin proteinof the invention or homologs thereof. Any characteristic epitopeassociated with a modified leukotoxin protein or its homolog may providethe requisite antigenic determinant. It is contemplated that monoclonalantibodies produced by the hybridoma cell lines fall within the scope ofthe present invention so long as they remain capable of selectivelyreacting with peptides from the modified leukotoxin protein or itshomolog. Monoclonal antibodies are useful in purification, usingimmunoaffinity techniques, of the proteins of the invention which theyare directed against.

III. Therapeutic Applications

As mentioned previously, the modified leukotoxin proteins of theinvention are useful as vaccines as the modified leukotoxin does notinsert into target cells and does not display toxic activity.Accordingly, a modified leukotoxin gene or protein of the invention maybe used to treat and prevent diseases caused bacteria that release thefull length or naturally occurring leukotoxin or related toxins. As anexample, a modified leukotoxin protein derived from a Mannheimialeukotoxin may be used to treat diseases or conditions caused byMannheimia infections such as respiratory disease. The modifiedleukotoxin derived from Mannheimia may also be useful in treatingdiseases or conditions caused by other bacteria when the modifiedleukotoxin shares epitopes that would cross react with the leukotoxinfrom the other bacteria.

Accordingly, the present invention provides a method of treating orpreventing a condition associated with a leukotoxin, such as aMannheimia haemolytica infection, comprising administering an effectiveamount of a modified leukotoxin gene or protein to an animal in needthereof. The invention also includes a use an effective amount of themodified leukotoxin gene or protein to treat or prevent a conditionassociated with a leukotoxin. The invention further includes a use of amodified leukotoxin gene or protein to prepare a medicament to treat orprevent a condition associated with a leukotoxin.

Administration of an “effective amount” of a modified leukotoxin gene orprotein of the present invention is defined as an amount of the gene orprotein, at dosages and for periods of time necessary to achieve thedesired result. For example, an effective amount of a substance may varyaccording to factors such as disease state, age, sex, and weight of therecipient, and the ability of the substance to elicit a desired immuneresponse in the recipient animal. Dosage regima may be adjusted toprovide an optimum therapeutic response.

The term “animal” as used herein includes all members of the animalkingdom, preferably a ruminant, more preferably cattle.

A modified leukotoxin protein of the invention or a homolog, analog,derivative or fragment thereof may be administered as a vaccinecomposition to prevent or treat respiratory disease in an animal, inparticular, a ruminant. In particular, a protein of the invention may beused to prevent or ameliorate respiratory disease associated with aMannheimia species in an animal, preferably a ruminant.

Animals can be immunized with the compositions of the present inventionby administration of the modified leukotoxin protein or a homolog,analog, derivative or fragment thereof. Prior to immunization, it may bedesirable to increase the immunogenicity of the modified leukotoxinprotein. This can be accomplished in any one of several ways known tothose skilled in the art. For example, the protein may be administeredlinked to a carrier. For example, a fragment may be conjugated with amacromolecular carrier. Suitable carriers are typically large, slowlymetabolized macromolecules such as: proteins (for example, serumalbumins, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin and the like) polysaccharides (for example,sepharose, agarose, cellulose, cellulose beads and the like) polymericamino acids (for example, polyglutamate, polylysine and the like), aminoacid co-polymers and inactive virus particles.

The proteins may be used in their native form or their functional groupcontent may be modified by, for example, succinylation of lysineresidues or reaction with Cys-thiolactone. A sulfhydryl group may alsobe incorporated into the carrier or the protein, for example by reactionof amino functions with 2-iminothiolane or the N-hydroxysuccinimideester of 3-(4-dithiopyridyl) propionate. Suitable carriers may also bemodified to incorporate spacer arms (such as hexamethylene diamine orother bifunctional molecules of similar size) for attachment ofpeptides.

Other suitable carriers for the proteins of the invention include VP6polypeptides of rotaviruses, or functional fragments thereof asdisclosed in U.S. Pat. No. 5,071,651. Also useful is a fusion product ofa viral protein and the epitope of interest made by methods disclosed inU.S. Pat. No. 4,722,840. Still other suitable carriers include cells,such as lymphocytes, since presentation in this form mimics the naturalmode of presentation in the subject, which gives rise to the immunizedstate. Alternatively, the proteins of the present invention, orantigenic fragment or homolog thereof, may be coupled to erythrocytes,preferably the subject's own erythocytes. Methods of coupling peptidesto proteins or cells are known to those skilled in the art.

The modified leukotoxin protein, or homolog, analog, derivative orfragment thereof, may be administered alone or mixed with apharmaceutically acceptable vehicle or excipient. Typically vaccines areprepared as injectables, either as liquid solutions or suspensions.Solid forms suitable for solution or suspension in liquid vehicles priorto injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.The active immunogenic ingredient is often mixed with vehiclescontaining excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable vehicles are, forexample, water, saline, dextrose, glycerol, ethanol, or the like, andcombinations thereof. In addition, if desired, the vehicle may containminor amounts of auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, or adjuvants which enhance theeffectiveness of the vaccine. Adjuvants may include, for example,muramyl dipeptides, avridine, aluminum hydroxide, oils, saponins andother substances known in the art. The preparation of such dosage formsis well known to those skilled in the art (see for e.g. Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. USA,1985).

Additional vaccine formulations which are suitable for other modes ofadministration include suppositories and, in some cases, aerosol,intranasal and oral formulations. For suppositories, the vehiclecomposition will include traditional binders and carriers, such aspolyalkaline glycols or triglycerides. Such suppositories may be formedfrom mixtures of active ingredient in the range of about 0.5% to about10% (w/w), preferably about 1% to about 2%. Oral vehicles include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium, stearate, sodium saccharincellulose, magnesium carbonate and the like. These oral vaccinecompositions may be taken in the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders andcontain from about 10% to about 95%, preferably about 25% to about 70%,of the active ingredient.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject protein by the nasal mucosa.

The modified leukotoxin proteins or homologs, analogs, derivatives orfragments thereof, may be formulated into vaccine compositions in eitherneutral or salt forms. Pharmaceutically acceptable salts include acidaddition salts formed with inorganic acids, such as, for example,hydrochloric or phosphoric acids, or organic acids, such as, forexample, acetic, oxalic, tartaric, mandelic acids and the like. Basicaddition salts may also be formed from free carboxyl groups and may bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium or ferric hydroxides, or organic bases such as, forexample, isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine,procaine and the like.

To immunize the subject, the protein of interest, or homologs, analogs,derivatives or fragments thereof, may be administered parenterally,usually by intramuscular injection in an appropriate vehicle, asdescribed above. Other modes of administration, such as subcutaneous,intravenous and intranasal delivery, are also acceptable. Injectablevaccine formulations will contain an effective amount of the activeingredient in a vehicle, the exact amount being readily determined by aperson skilled in the art. The active ingredient may typically rangefrom about 1% to about 95% (w/w) of the composition, or even higher orlower, if appropriate. The quantity to be administered depends on theanimal to be treated, the capacity of the animal's immune system tosynthesize antibodies, and the degree of protection desired. Effectivedosages can readily be established by one skilled in the art throughroutine trials establishing dose response curves. The subject isimmunized by administration of the antigenic protein, or homolog,analogs, derivatives or fragments thereof, in at least one dose, andpreferably two doses. Moreover, the subject may be administered as manydoses as is required to maintain a state of immunity to the respiratorydisease.

It is envisaged that the modified leukotoxin proteins of the invention,or homologs, analogs, derivatives or fragments thereof, may be used in acombination vaccine. For example, vaccines comprising proteins orpolyeptides expressing other antigens of M. haemolytica (such as thosedescribed in U.S. Pat. No. 5,871,750, incorporated herein by reference)or proteins or polypeptides expressing antigens of other diseasesaffecting the animal.

As another aspect of the present invention, vaccine compositions areprovided comprising a pharmaceutically acceptable carrier and a nucleicacid sequence encoding the modified leukotoxin protein, wherein themodification comprises the removal of nucleic acid sequences encodingamino acids within the hydrophobic transmembrane domains of the fulllength leukotoxin protein, or a nucleic acid sequence substantiallyhomologous and functionally equivalent thereto. The nucleic acidsequence is operably linked to regulatory sequences and inserted into asuitable vector so that it is capable of being expressed in vivo in theanimal. The insertion of the nucleic acid sequences of the inventioninto suitable vectors has been previously described herein. Suitablevectors for administering the nucleic acid as a vaccine includeretroviral vectors, adenoviral vectors and DNA virus vectors. Thevaccine vector containing a nucleic acid molecule of the invention caneither be (a) administered directly into an animal or (b) used totransform host cells in vitro and the transformed host cells can beadministered to an animal. For the former application, the vector may beadministered in admixture with suitable carriers as describedhereinabove for the leukotoxin protein vaccine. In a specificembodiment, the transformed host cell is a plant cell wherein thetransformed plant can be fed directly to animals for immunization.

The present invention will be further illustrated in the followingexamples. However, it is to be understood that these examples are forillustrative purposes only, and should not be used to limit the scope ofthe present invention in any manner.

EXAMPLES Example 1

The modified leukotoxin gene IktAΔN was prepared by deleting thehydrophobic domains of the full length M. haemolytica leukotoxin genecontaining nucleotides 618 to 1653. The nucleic acid sequence and aminoacid sequence of IktAΔN is shown in FIGS. 1A and 1B and 2A and 2B,respectively. The IktAΔN was cloned into an expression plasmid (pTTQ18)which placed the regulation of expression under the inducible tacpromoter. In the presence of another plasmid, pWAM716 which coded forthe hlyB/D secretion functions, the rLkt is secreted into thesupernatant of E. coli cultures for ease of recovery.

-   1. An E. coli strain that carries both plasmids, pLKTAN and pWAM716,    was used. As controls, E. coli carrying pWAM761 and pLKT60 (full    length IktA); and E. coli carrying pWAM716 and pTTQ18 (no iktA gene)    were also used as positive and negative controls respectively. All    strains were maintained on LT supplemented with ampicillin (100    μg/ml) and chloroamphenicol (25 μg/ml) to select for the plasmids.-   2. The following is the procedure for production and recovery of    rLkt from the E. coli cultures:    -   Step 1—Prepare overnight cultures in LT+ampicillin (100 μg/ml)        +chloramphenicol (25 μg/ml), 20 ml, 37° C.    -   Step 2—Subculture overnight into 1 lit.        LT+ampicillin+chloramphenicol (1/50), grow for 2 hr.    -   Step 3—Induce with IPTG, final conc. 0.5 mM, grow for 1 hr.    -   Step 4—Spin down cells in GSA rotor (12,000 rpm) 20 min.,        recover supernatant.    -   Step 5—Concentrate supernatant app. 10× using Amicon PM10        filtration apparatus.    -   Step 6—Dialyze concentrate against distilled water at 4° C. over        2 days with at least 5 changes of 10× vol. I used a membrane cut        off of app. 50 kDa.    -   Step 7—Lyophilize material, resuspend final powder in 2 ml        distilled water.-   3. The material prepared was injected into rabbits to produce    antibodies. The antibodies were tested in Western immunoblots as    well as toxin neutralization assays.-   4. Gels and Western immunoblots:

Samples: #3, full length Lkt-102

-   -   #4, Lkt-66    -   #5, negative sample, no IktA gene products    -   B122, M. haemolytica A1 total proteins.

FIG. 4—Coomassie stained SDS-PAGE, note the faint 102 kDa band in sample#3 and the thick 66 kDa band in sample #4.

FIG. 5—Western immunoblot of a duplicate set of protein preparationsimmunostained with rabbit anti-Lkt102 (#3) and anti-Lkt66 (#4).

The anti-Lkt102 (#3) serum recognized the 102 kDa and 66 kDa bands asexpected. The anti-66 (#4) serum also recognized the 102 kDa and the 66kDa bands, indicating that the 66 kDa antigen stimulated antibodyrespond against the full length toxin.

-   5. Toxin neutralization

Rabbits #35, 36 and 37 were immunized with 102 kDa toxin, all producedtoxin neutralization titers of 5 (½^(n)), the prebleed tirers are 1, 0.5and 2.

Rabbits #38 and 39 received the 66 kDa antigen. The toxin neutralizationtiters at the final bleed are 4 and 5 (prebleeds are 1 and 0)respectively. Therefore, the anti-66 serum also exhibit toxinneutralization activity similar to that of the anti-102.

Summary

A simple procedure for recovery of a modified leukotoxin (Lkt-66) fromthe culture supernatant of E. coli has been developed, wherein themodification comprises the removal of internal amino acids in thehydrophobic transmembrane domains of the full length leukotoxin protein.The yield of the modified protein was at least 50 times that of the fulllength leukotoxin rLkt-102 using the same procedure. The recoveredmaterials contain the 66 kDa modified Lkt which can simulate an immuneresponse in rabbits that recognize the authentic full length leukotoxinas well as toxin neutralization activity similar to that of the fulllength rLkt.

Example 2

1. Construction of IktA50

A derivative of Lkt66 (Lkt50) was made by further manipulation of theIktAΔN construct. PCR primers based on the IktA sequences at positions1355 and 2705 were designed to amplify a fragment of 1.35 kbp. Thisfragment codes for all of the antigenic regions of Lkt66, lacking theN-terminal portion of the protein. The removal of the N-terminal aminoacids of Lkt66 facilitates expression of the toxin derivative in plants.This construct, named Ikt50should produce a protein from amino acids 451to 901 of the full length leukotoxin, for a molecular mass of 49.6 kDa,slightly less than half of the full length leukotoxin of 102 kDa. Thecorresponding amino acid regions are:

-   phe-leu-leu-asn-leu-asn-lys-glu-leu-gln . . .    leu-ser-lys-val-val-asp-asn-tyr-glu-

Note that the last amino acid glu is continued into the GFP protein.

2. Cloning of Ikt50 into a Binary Vector

A binary vector pBINmgfp5-ER was used for cloning of the Ikt50fragmentand introduction into plants by Agrobacterium-mediated transformation.The vector is a derivative of the plant transformation vector pB1121(Clontech) and utilizes the cauliflower mosaic virus 35S promoter forexpression. It contains mgfp5-ER which codes for a variant of the greenfluorescent protein (GFP) for enhanced fluorescence (mGFP5). Inaddition, mgfp5-ER contains a signal peptide sequence and an endoplasmicreticulum (ER) retention sequence. PCR was carried out using specificprimers designed to introduce the appropriate restriction sites for thismanipulation. The PCR products were digested with the restriction enzymeand ligated into the plasmid, resulting in two constructs in which thepositions of Lkt50and mGFP5 were placed in front or after the other.These constructs should express a fusion protein of approximately 79kDa.

3. Transient Expression of Plasmid Constructs in Tobacco.

The two chimeric constructs, Lkt50-mGFP5 and mGFP5-Lkt50inserted intothe binary vectors were used to transform Agrobacterium. To rapidlyassess if these constructs were able to direct the production of thefusion proteins in plants, they were first transiently expressed intobacco by infiltration. Plasmid constructs containing promoterlessmgfp5-ER and 35S-driven mgfp5-ER were used as controls for transientexpression. Three to four days after infiltration, fluorescence wasobserved by microscopy only in plants injected with Agrobacteriumtransformed with 35S-mgfp5-ER. Plants infiltrated with Agrobacteriumcontaining the promoterless construct showed no fluorescence. Little orno fluorescence was observed in the infiltrated regions of plantsinjected with Agrobacterium containing either Lkt50construct. Theinfiltrated areas were excised and examined for the presence ofrecombinant fusion protein by Western immunoblot with rabbit anti-Lkt66antibodies (FIG. 6). An immunoreactive band of approximately 79 kDa waspresent only when the Lkt50-mGFP5 construct was used. The size of theband corresponded to that predicted from the nucleotide sequence of thefusion protein. No specific immunostaining was observed with the controlplasmids. Thus, it appeared that when mGFP5 was fused after Lkt50, thefusion was expressed in tobacco and resulted in the accumulation of asignificant amount of recombinant protein. This construct was selectedfor the production of transgenic white clover lines.

4. Transgenic White Clover Expressing Lkt50-mGFP5

Transgenic clover lines expressing mGFP5 and Lkt50-mGFP5 were producedby A. tumefaciens-mediated transformation. PCR was used to confirm thatthe transgenes were present in transformed plants. By conventionalfluorescence microscopy, mGFP5 fluorescence was easily detected in mGFP5expressing plants. Consistent with the results obtained with transientexpression, little to no fluorescence was observed in Lkt50-mGFP5transformed plants. However, when these plants were further investigatedusing laser scanning confocal microscopy, green fluorescence wasdetected in clover transformed with both the mgfp5-ER and lkt50-mgfp5-ERconstructs (FIG. 7). As expected, mGFP5 fluorescence was more intensethan that observed for Lkt50-mGFP5. The pattern of green fluorescenceobserved in clover leaves was consistent with a localization of therecombinant protein in the endoplasmic reticulum. The cells containedlarge vacoules which resulted in a distribution of fluorescence aroundthe cell pheriphery. The fusion protein exhibited a perinuclearlocalization and was clearly excluded from the nucleus. A characteristicreticulate network was seen in some cells when the appropriate plane offocus was used.

Expression of a recombinant fusion protein containing both Lkt66 andmGFP5 epitopes were confirmed by Western immunoblot analysis (FIGS. 8A &B). Both rabbit anti-Lkt66 and rabbit anti-GFP (Clontech) antibodiesrecognized a protein migrating at approximately 79 kDa. Preliminaryscanning densitometric analysis of gels and the blots from one of theLkt50-mGFP5 expressing clover lines (LKT6) suggested that therecombinant fusion protein constitute approximately 1% of the solubleproteins extracted from transgenic clover.

5. Stability of Lkt50-mGFP5

The stability of the Lkt50-mGFP5 fusion protein in harvested plants wasexamined. Transgenic clover expressing Lkt50-mGFP5 was harvested andallowed to dry at ambient temperatures. Protein extracts were preparedfrom plant material at different stages of drying and analyzed byWestern immunoblot. After 4 days of drying, there did not appear to besignificant degradation of the fusion protein as no lower molecularweight immunoreactive species was observed (FIG. 8C).

6. Immunogenicity of Lkt50-mGFP5 Produced From Transgenic White Clover

To determine if Lkt50-mGFP5 produced by clover was able to elicit animmune response, rabbits were immunized with either a saline extract ora Lkt50-mGFP5-enriched chromatographic fraction prepared from cloverleaves.

The Lkt50-mGFP5-enriched fractions were produced by chromatofocusing(Pharmacia). A soluble protein extract prepared from transgenic cloverwas applied to a PBE 94 column and resulting fractions were analyzed byWestern immunoblot (FIG. 9). Most of the fusion protein eluted infractions 6 to 8 and these fractions were pooled for rabbitimmunization. The fusion protein could be partially separated fromribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the mostabundant protein in plant tissue, most of which eluted in fractions 5and 6. Under the conditions used in the fractionation, Lkt50-mGFP5 wasstable to degredation as indicated by the absence of lower molecularweight immunoreactive bands in the column fractions.

Sera were obtained from both pre-immunized and immunized rabbits andtested for the presence of antibodies by Western immunoblot (FIG. 10).All rabbits receiving fractions containing Lkt50-mGFP5 as antigen wereable to produce antibodies directed against the authentic Lkt from M.haemolytica A1 (FIG. 10, lanes 3-6). Sera from mock-immunized rabbits(FIG. 10, lanes 1-2) or rabbits immunized with wild type white cloverextract failed to immunostain Lkt (data not shown). Preimmune sera fromall the rabbits also did not detect Lkt (data not shown).

Toxin neutralization assay was performed to determine if neutralizingantibodies were present in immune sera. All rabbits immunized withLkt50-mGFP5 extracts as antigen exhibited neutralizing titres of up to adilution of 1/16 (Table 1). Sera from mock-immunized rabbits (Table 1),from rabbits immunized with wild type white clover extract or preimmunesera) failed to neutralize at the lowest dilutions (data not shown).

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Neutralizing titres of sera from rabbits immunized with rLkt-GFPfusion protein. Rabbit Neutralizing Titre^(a) Immunogen No. Pre-immune2^(nd) bleed 3^(rd) bleed 4^(th) bleed Lkt50-mGFP5 41 0 0 4 3.5 (salineextract) 42 0 0 1 1 Lkt-mGFP5 43 0 0 1 1 (column fraction) 44 0 0 1.5 1Mock 45 0 0 0 0 46 0 0 0 0 ^(a)Values are mean reciprocal log₂ serumdilutions giving at least 50% neutralization of toxicity

1. An isolated nucleic acid molecule which a) encodes a modifiedleukotoxin protein with an amino acid sequence consisting of thesequence SEQ ID NO:4, or b) having a nucleic acid sequence consisting ofthe sequence of SEQ ID NO:3.
 2. A nucleic acid construct comprising: (a)a nucleic acid molecule of claim 1; and (b) control sequences that areoperably linked to the nucleic acid sequence whereby the nucleic acidsequence can be transcribed and translated in a host cell.
 3. Anisolated host cell transformed by a nucleic acid construct according toclaim
 2. 4. An isolated host cell according to claim 3 wherein said hostcell is a bacterium.
 5. An isolated host cell according to claim 3wherein said host cell is a plant cell.
 6. An immunogenic compositioncomprising: (a) a nucleic acid sequence according to claim 1; and (b) apharmaceutically acceptable carrier.
 7. An immunogenic compositioncomprising: (a) the DNA construct according to claim 2; and (b) apharmaceutically acceptable carrier.